A novel angiotensin analog with subnanomolar affinity for angiotensin- converting enzyme

This study demonstrates that a novel angiotensin I analog, angiotensinogen 3-11(Lys11), possesses a high affinity for angiotensin- converting enzyme (ACE), which is substantially greater than the endogenous substrates. This assessment is based on data derived from a variety of techniques. First, the binding characteristics of 125I-angiotensinogen 3- 11(Lys11) were examined. Equilibrium saturation isotherms utilizing guinea pig lung membranes revealed that 125I-angiotensinogen 3-11 (Lys11) bound a single high-affinity site in the presence of EDTA exhibiting a K(d) of 0.15 ± 0.02 nM with a B(max) = 4295 ± 535 fmol/mg of protein. Competition studies revealed the following rank order of binding affinity: 125I- angiotensinogen 3-11(Lys11) >> bradykinin >> angiotensin I. Next, SDS- polyacrylamide gel electrophoresis analysis revealed that chemically cross- linked 125I-angiotensinogen 3-11 (Lys11) specifically bound a protein of M(r) 173,000 that had the same molecular weight as ACE. Utilizing in vitro autoradiography, the binding distributions of 125I-angiotensinogen 3- 11(Lys11) and the ACE inhibitor, 125I-351A, were also compared. These experiments demonstrated that the binding distributions of 125I- angiotensinogen 3-11(Lys11) and 125I-351A are identical in the guinea pig lung and testes. Finally, the purification of ACE from guinea pig serum was monitored with 125I-angiotensinogen 3-11(Lys11) and 125I-351A binding. These results demonstrated that the binding site for 125I- angiotensinogen 3-11(Lys11) and 125I-351A copurified. These experiments indicate that the novel angiotensin I analog, 125I-angiotensinogen 3-11 (Lys11) binds to ACE and suggest that there are critical binding sites outside the catalytic domains of ACE that determine binding specificity and affinity.